Photodynamic therapy (PDT), a unique antitumor modality involving a sensitizing agent, photoexciting light and molecular oxygen, is characterized by local generation of singlet oxygen and other cytotoxic oxidants. When subjected to PDT-induced oxidative stress, many tumors succumb to apoptotic cell death, and much has been learned about how this is affected by factors such as sensitizer localization and efficiency of toxic oxidant generation. However, the influence of metabolic and environmental factors, is still not well understood. Studies supported by the existing grant have focused largely on the effects of nitric oxide (NO) in this regard. Nitric oxide synthase (NOS)-generated NO in low doses is known to have pro-survival and growth-promoting effects on various tumors. Using in vitro models of 5- aminolevulinic acid (ALA)-based PDT and chemical NO donors, we have shown that NO can protect tumor cells against necrotic photokilling by either scavenging lipid-derived radicals or by signaling for heme oxygenase-1 and ferritin induction, leading to depletion of prooxidant iron. We recently discovered that NO is overproduced by ALA/light-stressed breast tumor cells due to rapid and prolonged upregulation of inducible NOS and that this substantially increases cell resistance to intrinsic apoptotic photokilling. This proposal developed largely from this novel observation and is based on the following hypothesis: Under PDT stress, many tumors will overexpress NOS and NO as a cytoprotective response, and this can compromise PDT efficacy. Our overall plan for testing this hypothesis is to study: (i) relative abilities of various established breast, prostate, and skin carcinoma cells to overexpress cytoprotective NOS/NO under ALA/light stress;(ii) mechanisms of NOS induction by photostress;(iii) cytoprotective mechanisms of stress-induced NO;(iv) effects of this NO on bystander cells;and (v) PDT induction of NOS/NO in a mouse xenograft model and NOS inhibitor effects. Planned methods include: cultured cell sensitization, irradiation, and apoptosis evaluation;use of NOS inhibitors, NO scavengers, and chemical NO donors;RNA interference;immunoblotting;confocal microscopy;and PDT of human tumors implanted in immunosuppressed mice;in addition to ALA-PDT, classical Photofrin-PDT will be used. This proposal is significant and innovative for the following reasons: (i) Although positive effects of NOS inhibitors in animal tumor PDT have been reported, there is no known evidence for endogenous NOS/NO upregulation due to PDT;(ii) The prospect of eventually using NOS inhibitors to improve clinical PDT outcomes is favorable, given that human testing of at least one of those to be studied, GW274150 (as an anti-asthmatic), has been reported. PUBLIC HEALTH RELEVANCE: Relevance to Public Health Photodynamic therapy (PDT) is a unique approach for treating cancer in which a sensitizing agent, visible light, and molecular oxygen interact to destroy tumor cells and prevent further tumor growth. Nitric oxide (NO), a small gaseous molecule produced naturally by nitric oxide synthase (NOS) enzymes, has a variety of biological functions, including the ability to promote growth and redistribution of tumor cells. We in this laboratory recently discovered that cultured human breast tumor cells rapidly overproduce inducible NOS (iNOS) and NO when given a PDT-like challenge, and that this makes the cells more resistant to photokilling. The major goal of this project is to elucidate the mechanism of iNOS induction under photostress and how the resulting NO acts cytoprotectively. Studying the ability of iNOS inhibitors to promote tumor cell killing in vitro and in animal models is a crucial part of the project because future clinical use of such inhibitors could dramatically improve PDT effectiveness in cancer treatment.